Polypeptide and use thereof for regulating blood glucose and / or reducing fat

Abstract

The invention discloses a polypeptide and application thereof in regulating blood sugar and / or reducing fat, and the amino acid sequence of the polypeptide comprises any one of SEQ ID No.1 to SEQ ID No.4 or a sequence obtained by modifying at least one amino acid in the SEQ ID No.1 to SEQ ID No.4. The polypeptide disclosed by the invention has physiological activity of promoting fat metabolism and regulating blood sugar, so that the effect of treating or preventing diseases related to blood sugar imbalance or fat metabolism imbalance can be effectively achieved by adding an effective amount of the polypeptide disclosed by the invention or the composition containing the polypeptide to an individual.

Description

Technical Field

[0001] The present invention relates to a polypeptide and its use, and in particular to a polypeptide and its use for regulating blood sugar and / or reducing fat. Background Art

[0002] According to the survey, the prevalence of diabetes among residents over 18 years old in Taiwan, China continues to rise, even higher than the global average prevalence of diabetes, and diabetes has a trend of becoming younger. In addition to genetics, the cause of diabetes is mostly caused by refined and high-sugar and high-fat eating habits. Since eating habits are difficult to change, many nutritional supplements related to blood sugar regulation or weight loss have appeared on the market, among which the main active ingredient of many nutritional supplements is peptides.

[0003] Peptides are composed of amino acids. Since peptides are substances between amino acids and proteins, their molecular weight is significantly lower than that of proteins. Therefore, they can be directly absorbed or have a better absorption rate after entering the human body. Recent studies have found that many peptides have specific physiological functions or activities, such as preventing osteoporosis, inhibiting cancer, and anti-aging. Therefore, peptides are currently widely used in nutritional supplements or skin care products. At present, most methods of obtaining peptides are to hydrolyze proteins with enzymes. Although the enzyme hydrolysis method can quickly obtain peptides, it is a big problem to control quality and improve purity. Summary of the invention

[0004] The main purpose of the present invention is to provide a polypeptide and its use for regulating blood sugar and / or reducing fat. Specifically, the polypeptide disclosed in the present invention has the ability to activate insulin receptors, promote cells to actively transport glucose, thereby regulating blood sugar, and promote cellular fat oxidation metabolism, thereby effectively achieving the effect of regulating blood sugar and / or inhibiting fat accumulation.

[0005] A secondary purpose of the present invention is to provide a polypeptide and its use for regulating blood sugar and / or reducing fat. Based on the fact that the polypeptide disclosed in the present invention has the effects of regulating blood sugar and promoting fat metabolism at the same time, the polypeptide disclosed in the present invention has the ability to regulate multiple metabolic indicators and can effectively achieve the effect of preventing or treating metabolic syndrome.

[0006] To achieve the above object, the amino acid sequence of the polypeptide disclosed in the present invention comprises a sequence shown in SEQ ID No.1, a sequence shown in SEQ ID No.2, a sequence shown in SEQ ID No.3, a sequence shown in SEQ ID No.4, or a sequence obtained by modifying one or more amino acids in any of the above sequences.

[0007] In one embodiment of the present invention, the amino acid sequence of the polypeptide is SEQ ID No.1.

[0008] In the next embodiment of the present invention, the amino acid sequence of the polypeptide is SEQ ID No.2.

[0009] In another embodiment of the present invention, the amino acid sequence of the polypeptide is SEQ ID No.3.

[0010] In another embodiment of the present invention, the amino acid sequence of the polypeptide is SEQ ID No.4.

[0011] The polypeptide disclosed in the present invention can be obtained by artificial synthesis, enzyme hydrolysis, solvent extraction or other protein preparation techniques known in the art. For example, the polypeptide with the amino acid sequence of SEQ ID No. 4 can be prepared by artificial synthesis, or by subjecting the polypeptide with the amino acid sequence of SEQ ID No. 3 to an enzyme hydrolysis procedure.

[0012] Furthermore, since the polypeptide disclosed in the present invention has the function of regulating blood sugar and / or promoting fat metabolism, in one embodiment of the present invention, the polypeptide disclosed in the present invention is used to prepare a composition for regulating blood sugar, that is, when an effective amount of the polypeptide disclosed in the present invention is administered to an individual with blood sugar regulation symptoms or a disease related to blood sugar metabolism imbalance, the polypeptide disclosed in the present invention can activate insulin receptors and promote active glucose transport in cells, thereby effectively achieving the effect of regulating individual blood sugar and delaying the occurrence of blood sugar-related diseases.

[0013] The blood sugar regulating composition disclosed in the present invention comprises a polypeptide having an amino acid sequence of SEQ ID No. 1, a polypeptide having SEQ ID No. 2 and a polypeptide having SEQ ID No. 3.

[0014] Among them, the blood sugar regulating composition disclosed in the present invention comprises a polypeptide with an amino acid sequence of SEQ ID No. 1, a polypeptide with SEQ ID No. 2, a polypeptide with SEQ ID No. 3 and a polypeptide with SEQ ID No. 4.

[0015] In another embodiment of the present invention, the polypeptide disclosed in the present invention is used to prepare a fat-reducing composition to enhance the activity of individual cells in metabolizing fat, so that the individual can still inhibit the formation of body fat and fat accumulation in the body under the environment of high-fat or high-calorie diet, thereby effectively achieving the effect of reducing fat and weight.

[0016] The fat-reducing composition disclosed in the present invention comprises a polypeptide having an amino acid sequence of SEQ ID No. 1, a polypeptide having SEQ ID No. 2 and a polypeptide having SEQ ID No. 3.

[0017] The fat-reducing composition disclosed in the present invention comprises a polypeptide having an amino acid sequence of SEQ ID No. 1, a polypeptide having an amino acid sequence of SEQ ID No. 2, a polypeptide having an amino acid sequence of SEQ ID No. 3, and a polypeptide having an amino acid sequence of SEQ ID No. 4.

[0018] Another embodiment of the present invention discloses a composition comprising an effective amount of the above-mentioned polypeptide, wherein the composition can be a medicine, a food, a nutritional supplement, etc. By administering the composition to a subject, the effect of treating or preventing diseases caused by blood sugar imbalance or fat metabolism imbalance can be achieved, wherein the diseases caused by blood sugar imbalance or fat metabolism imbalance include hyperglycemia, obesity, diabetes, metabolic syndrome, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the LC spectrum of the polypeptide extract disclosed in Example 1 of the present invention. DETAILED DESCRIPTION

[0020] The present invention discloses a polypeptide for regulating blood sugar and / or reducing fat, wherein the amino acid sequence thereof comprises any one of SEQ ID No.1 to SEQ ID No.4 (as shown in Table 1 below), or any one of SEQ ID No.1 to SEQ ID No.4 with one or more amino acids modified and having 90% homology. Since the polypeptide disclosed in the present invention has the functions of activating insulin receptors, promoting active glucose transport in cells, and promoting cellular fat oxidation metabolism, when an effective amount of the polypeptide disclosed in the present invention or a composition containing the polypeptide is administered to an individual, the effect of reducing fat and / or regulating blood sugar can be effectively achieved, thereby preventing or treating metabolic syndrome, hyperglycemia, diabetes, and other diseases related to abnormal blood sugar or fat metabolism.

[0021] Table 1: Amino acid sequences of the polypeptides disclosed in the present invention

[0022] Peptide Name Amino acid sequence number Amino acid sequence Polypeptide 1 SEQ ID No.1 GGGDATRYSTGGGYS Polypeptide 2 SEQ ID No.2 INPNEQREV Polypeptide 3 SEQ ID No.3 MQIFVKTLTG Polypeptide 4 SEQ ID No.4 MQIFVK

[0023] The individual may be a dog, a cat, a cow, a pig, a human or any other animal.

[0024] In one embodiment of the present invention, the amino acid sequence of the polypeptide for regulating blood sugar and / or reducing fat disclosed in the present invention is SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 or SEQ ID No.4.

[0025] The term "modification" in the present invention refers to substitution, deletion or insertion of at least one amino acid site in an amino acid sequence.

[0026] The so-called "polypeptide" in the present invention refers to a small molecule protein composed of multiple amino acids.

[0027] The polypeptide disclosed in the present invention can be prepared by artificial synthesis, enzyme hydrolysis and other well-known peptide obtaining techniques in the art. The so-called artificial synthesis method includes chemical synthesis, biological synthesis and other methods. Among them, the chemical synthesis method connects multiple amino acids through peptide bonds to obtain the target peptide, and can be further divided into liquid phase synthesis, solid phase synthesis and the like according to the synthesis technology; and the biological synthesis method is to produce the target peptide through recombinant microorganisms or recombinant cells, and the nucleic acid molecule that can express the target peptide is transferred into a microorganism or a cell through biotechnology in the recombinant microorganism or recombinant cell.

[0028] The polypeptides disclosed in the present invention exist in animals or plants, and thus can be separated from raw materials by extraction or other separation techniques known in the art, such as the peel, pulp, fruit, seeds, and leaves of pumpkin, watermelon, cucumber, loofah, or other Cucurbitaceae plants.

[0029] The polypeptide disclosed in the present invention can also be purified from plant protein or animal protein, such as pumpkin protein, loofah protein, cucumber protein or other proteins from Cucurbitaceae plants, and the commonly used protein purification techniques in the prior art include salting out, colloid filtration, chromatography, etc. For example, before protein purification, the plant protein or animal protein can be subjected to enzyme hydrolysis or extraction, and then the target peptide can be obtained through the above-mentioned protein purification technology.

[0030] The "composition" disclosed in the present invention refers to a substance containing an effective amount of the polypeptide disclosed in the present invention, such as nutritional supplements, pharmaceuticals, foods, feeds, animal nutrition products, etc., wherein the so-called effective amount is 0.01%-100% of the total amount of the composition. The dosage form of the composition includes but is not limited to powders, tablets, drops, liquids, etc., and the composition may further include at least one group of components, such as the composition includes the polypeptide disclosed in the present invention and at least one pharmaceutically acceptable carrier; or the composition includes the polypeptide disclosed in the present invention and at least one food acceptable component, etc.

[0031] In order to illustrate the technical features and effects of the present invention, several embodiments are given below with accompanying data.

[0032] The polypeptides used in the following examples were obtained by artificial synthesis, and the sequences were confirmed to be correct by HPLC and LC / MASS identification. There are 5 polypeptides used in the following examples, and their amino acid sequences are shown in SEQ ID No.1 to SEQ ID No.5, respectively. The molecular weight of each polypeptide is shown in Table 2 below.

[0033] Table 2: Molecular weight of each polypeptide

[0034] Peptide Name Amino acid sequence number Molecular weight Amino acid sequence Polypeptide 1 SEQ ID No.1 1405.40 GGGDATRYSTGGGYS Polypeptide 2 SEQ ID No.2 1098.53 INPNEQREV Polypeptide 3 SEQ ID No.3 1404.59 MQIFVKTLTG Polypeptide 4 SEQ ID No.4 765.62 MQIFVK Polypeptide 5 SEQ ID No.5 841.53 VATVIQ

[0035] The cells used in the following examples are all commercial cell lines, so no deposit is required.

[0036] Example 1: Preparation of Polypeptide Extract

[0037] Pumpkin seeds are taken, and after pretreatment procedures such as washing and grinding, an extraction procedure is performed. The extraction procedure can be an extraction technique known to those skilled in the art, such as solvent extraction, supercritical extraction, ultrasonic extraction, etc., and in the operation of this embodiment, carbon dioxide is used as a solvent, and the extraction procedure is performed by supercritical extraction, wherein the extraction temperature is 30-80°C and the pressure is 8.2-50.0Mpa; after the extraction is completed, a crude extraction product is obtained, and then disinfection, drying, powderization and other procedures are performed to obtain the polypeptide extract disclosed in the present invention, and its LC spectrum is as follows Figure 1 shown.

[0038] Depend on Figure 1It can be seen from the content that the polypeptide extract disclosed in the present invention has peaks at retention times of 8.1-8.2, 9.4-9.5, 13.0-13.1, 14.5-14.6, 16.2-16.3, 17.9-18.0, 19.6-19.7, 20.1-20.2, 22.0-22.1, and 22.6-22.7 minutes, indicating that the polypeptide extract disclosed in the present invention is indeed a mixture of multiple peptides. Specifically, the retention time of peptide fragment 1 (P fraction 1) is 8.160 minutes; the retention time of peptide fragment 2 (P fraction 2) is 9.419 minutes; the retention time of peptide fragment 3 (P fraction 3) is 13.025 minutes; the retention time of peptide fragment 4 (P fraction 4) is 14.589 minutes; the retention time of peptide fragment 5 (P fraction 5) is 16.270 minutes; the retention time of peptide fragment 6 (P fraction 6) is 17.902 minutes; the retention time of peptide fragment 7 (P fraction 7) is 19.112 minutes; the retention time of peptide fragment 8 (P fraction 8) is 19.626 minutes; the retention time of peptide fragment 9 (P fraction 9) is 20.150 minutes; the retention time of peptide fragment 10 (P fraction 10) is 22.059 minutes; the retention time of peptide fragment 11 (P fraction The retention time of peptide fragment 11) was 22.672 minutes; the retention time of peptide fragment 19 (P fraction 19) was 23.892 minutes.

[0039] Example 2: Animal Experiment (I)

[0040] A number of 8-week-old SD rats were randomly divided into two groups, and the type 1 diabetes model was induced by intraperitoneal injection of the drug STZ (Streptozotocin) (55-75 mg / kg). One week after induction, fasting blood was collected to test the blood glucose level. If it exceeded 230 mg / dl, the induction was considered successful, and the injection of the drug STZ was stopped. The rats in the blank group were fed a high-calorie diet after the injection of the drug STZ. The rats in the polypeptide group were fed a high-calorie diet and the polypeptide extract disclosed in Example 1 after the injection of the drug STZ, wherein the dosage of the polypeptide was converted from the human dosage of 300 mg / day, which means that the rat dosage was 31.2 mg / kg (the calculation formula is 300 / 60*6.25).

[0041] After the rats in each group were raised under the above conditions for 8 weeks, their blood glucose levels and glycosylated hemoglobin (HbA1c) were tested. The results are shown in Table 3 below.

[0042] From the results in Table 3, it can be seen that the blood sugar level of the rats in the polypeptide group is significantly lower than that of the rats in the blank group, with a decrease of about 40%; and the glycosylated hemoglobin of the rats in the polypeptide group is lower than that of the rats in the blank group. The results show that the polypeptide extract disclosed in the present invention can effectively regulate the blood sugar level of an individual, and can effectively reduce the possibility of an individual developing diabetes even in the case of damaged pancreatic islets.

[0043] Table 3: Blood glucose levels and glycosylated hemoglobin in each group of rats after the experiment

[0044] Group Blood glucose level (mg / dL) Glycated hemoglobin (%) Blank group 259±18.1 9.7±2.6 Polypeptide group 170±18.4 10.2±1.1

[0045] Example 3: Animal Experiment (II)

[0046] Several 8-week-old SD rats were randomly divided into two groups and the drug STZ (Streptozotocin) was intraperitoneally injected at a low dose once every three days to induce type 2 diabetes. After induction, fasting blood samples were collected every week to test blood glucose levels. When the blood glucose level exceeded 230 mg / dl, the induction was considered successful and the injection of the drug STZ was stopped.

[0047] After the injection of each group of rats, they were fed with high-calorie feed at the same time, and one group of rats (hereinafter referred to as the polypeptide group) was administered with the polypeptide extract disclosed in Example 1 at the same time, wherein the dosage of the polypeptide extract was converted from the human dosage of 300 mg / day, which means that the rat dosage was 31.2 mg / kg (calculated as 300 / 60*6.25). After each group of rats was fed under the above conditions for 8 weeks, their blood sugar levels and glycosylated hemoglobin (HbA1c) were tested, and the results are shown in Table 4 below.

[0048] From the results in Table 4, it can be seen that the blood sugar level of the rats in the polypeptide group is significantly lower than that of the rats in the blank group, with a decrease of about 38.8%; and the glycosylated hemoglobin of the rats in the polypeptide group is lower than that of the rats in the blank group, with a decrease of about 14.3%. These results show that when an individual suffers from type 2 diabetes or has a high risk of suffering from type 2 diabetes, the administration of the polypeptide extract disclosed in the present invention can effectively regulate the individual's blood sugar level, thereby preventing or delaying the occurrence of diabetes or diseases related to blood sugar metabolism imbalance.

[0049] Table 4: Blood glucose levels and glycosylated hemoglobin in each group of rats after the experiment

[0050] Group Blood glucose level (mg / dL) Glycated hemoglobin (%) Blank group 222±30.9 9.28±1.8 Polypeptide group 145±14.6 7.95±0.9

[0051] Example 4: Animal Experiment (III)

[0052] A number of 8-week-old SD rats were randomly divided into two groups. The rats in the blank group were only fed with high-calorie feed, and the rats in the polypeptide group were fed with high-calorie feed and the polypeptide extract disclosed in Example 1 at the same time. The dosage of the polypeptide extract was converted from the human dosage of 300 mg / day, which means that the dosage of the rats was 31.2 mg / kg (calculated as 300 / 60*6.25).

[0053] After the rats in each group were raised under the above conditions for 8 weeks, their body weights were measured, and after they were sacrificed, their abdominal fat weights were measured. The results are shown in Table 5 below.

[0054] From the results in Table 5, it can be seen that the body weight and abdominal fat weight of the rats in the polypeptide group were significantly lower than those of the rats in the blank group. Specifically, the body weight and abdominal fat weight of the rats in the polypeptide group were reduced by 17.9% and 27.7% respectively compared with those of the rats in the blank group.

[0055] The results show that when individuals continue to eat a high-fat or high-calorie diet and administer the polysaccharide extract disclosed in the present invention, it can not only inhibit fat accumulation, but also increase fat metabolism to achieve the effect of weight loss, reducing body fat or avoiding a significant increase in weight. In other words, the polysaccharide extract peptide disclosed in the present invention can be used to prevent and / or treat diseases related to fat metabolism, such as obesity, metabolic syndrome, etc.

[0056] Table 5: Body weight and abdominal fat of rats in each group after the experiment

[0057] Group Weight (g) Belly fat (g) Blank group 648.4±79.08 38.92±9.57 Polypeptide group 532.6±89.23 28.12±10.12

[0058] Example 5: Cell test

[0059] From the results of the above examples, it can be seen that the polypeptide extract disclosed in Example 1 has the activities of reducing glycosylated hemoglobin, regulating blood sugar, and increasing fat metabolism. Therefore, the polypeptides shown in SEQ ID No.1 to SEQ ID No.5 were separated and sequenced, and prepared by artificial synthesis to carry out the following cell experiments.

[0060] Mouse myoblast C2C12 was taken and cultured in groups for 24 hours. Human insulin (MedChemExpress), polypeptide 1 disclosed in the present invention (SEQ ID No. 1), polypeptide 2 disclosed in the present invention (SEQ ID No. 2), polypeptide 3 disclosed in the present invention (SEQ ID No. 3), polypeptide 4 disclosed in the present invention (SEQ ID No. 4) and polypeptide 5 (amino acid sequence coded as SEQ ID No. 5) were added to the culture medium respectively, so that the final concentration of the culture medium was 20 μL / mL, and each cell group was cultured for another 24 hours. The culture medium of each cell group was removed, and after washing with phosphate buffer, RIPA buffer to inhibit enzyme was added, and the reaction was refrigerated for 30 minutes. After thawing, the supernatant of each cell group was obtained by centrifugation, and then analyzed by Western blot to obtain the expression changes of p-IR / IR, p-Akt, GLUT4, mTOR, PPARα and p-AMPK in each cell group. The results are shown in Table 6 below.

[0061] Table 6: Results of detecting changes in protein expression related to blood glucose regulation, insulin receptor activation, and fat oxidation metabolism in each cell group.

[0062] Cell treatment conditions p-IR / IR p-Akt GLUT4 PGC-1α mTOR PPARα p-AMPK Added Polypeptide 1 1.81 +196％ +376％ +161％ +136％ +173％ +232％ Added Polypeptide 2 1 +29％ +23％ +0％ +22％ +62％ +70％ Added Polypeptide 3 3.08 +153％ +582％ +258％ +117％ +196％ +210％ Added Polypeptide 4 1.97 -3％ +156％ +155％ +74％ +151％ +91％ Added Polypeptide 5 1 -42％ +34％ +3％ +0 +23％ +7％ Add insulin 1.23 +56％ +41％ +50％ -2％ +40％ +1％

[0063] From the results in Table 6, it can be seen that the polypeptide 1 with the amino acid sequence of SEQ ID No.1, the polypeptide 3 with the amino acid sequence of SEQ ID No.3 and the polypeptide 2 with the amino acid sequence of SEQ ID No.2 disclosed in the present invention can promote the phosphorylation of insulin receptor, increase the expression of phosphorylated AKT and GLUT4, and enhance the expression of key proteins PGC-1α and PPARα in the fat oxidation pathway without insulin induction; and the polypeptide 4 with the amino acid sequence of SEQ ID No.4 disclosed in the present invention, although it does not significantly enhance the expression of phosphorylated AKT, it can still promote the phosphorylation of insulin receptor, increase the expression of GLUT4, and enhance the expression of key proteins PGC-1α and PPARα in the fat oxidation pathway without insulin induction. Compared with the polypeptides with the amino acid sequences of SEQ ID No.1-SEQ ID No.4, the polypeptide 5 with the amino acid sequence of SEQ ID No.5 cannot enhance the expression of phosphorylated AKT, and the enhancement effect on the expression of GLUT4, PGC-1α and PPARα is poor. From the above results, it can be seen that the polypeptides with amino acid sequences of SEQ ID No. 1 to SEQ ID No. 4 disclosed in the present invention have better expression levels for p-IR / IR, p-Akt, GLUT4, mTOR, PPARα and p-AMPK than insulin.

[0064] The results in Table 6 show that the polypeptides with amino acid sequences of SEQ ID No. 1 to SEQ ID No. 4 disclosed in the present invention can activate insulin receptors, promote active glucose transport in cells to regulate blood sugar, and promote cellular fat oxidation metabolism, etc., and can be used to achieve the effect of regulating individual blood sugar or reducing body weight. In other words, by administering an effective amount of the polypeptides with amino acid sequences of SEQ ID No. 1 to SEQ ID No. 4 disclosed in the present invention or a composition containing any of the above polypeptides to an individual, it is possible to treat and / or prevent diseases related to blood sugar imbalance or abnormal fat metabolism, such as diabetes, hyperglycemia, obesity, metabolic syndrome, etc.

Claims

1. A polypeptide, characterized in that: The amino acid sequence of the polypeptide comprises a sequence shown in SEQ ID No.1, a sequence shown in SEQ ID No.2, a sequence shown in SEQ ID No.3, a sequence shown in SEQ ID No.4, or a sequence obtained by modifying one or more amino acids in any of the above sequences.

2. The polypeptide according to claim 1, characterized in that The amino acid sequence is selected from the group consisting of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.

4.

3. A composition, characterized in that The invention comprises an effective amount of a polypeptide, wherein the amino acid sequence of the polypeptide is selected from the group consisting of one or more of the following sequences: SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, and SEQ ID No.

4.

4. The composition according to claim 3, characterized in that The composition comprises the polypeptides shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.

4.

5. Use of the polypeptide according to claim 2 in preparing a composition for regulating blood sugar.

6. The use according to claim 5, characterized in that The composition comprises the polypeptides shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.

4.

7. Use of the polypeptide according to claim 2 in preparing a fat-reducing composition.

8. The use according to claim 7, characterized in that The composition comprises the polypeptides shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4.

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